Surface display of whole antibodies in eukaryotes

Abstract

Methods for display of recombinant whole immunoglobulins or immunoglobulin libraries on the surface of eukaryote host cells, including yeast and filamentous fungi, are described. The methods are useful for screening libraries of recombinant immunoglobulins in eukaryote host cells to identify immunoglobulins that are specific for an antigen of interest.

What is claimed is:  
     
       1. A method of producing yeast host cells that produce immunoglobulins having VH domains, that are fused to Fc domains, and VL domain that are fused to constant regions, and having antigen binding sites with binding specificity for antigens of interest, wherein N-glycans produced by the host cells comprise the structure Man 5 GlcNAc 2 , the method comprising:
 (a) providing a library of yeast host cells, wherein N-glycans produced by the host cells comprise the structure Man 5 GlcNAc 2 , wherein the yeast host cells of the library comprise nucleic acid molecules encoding a genetically diverse population of VL domains and VH domains; 
 wherein the VH domains are fused to Fc domains; 
 wherein the VL domains are fused to constant regions; 
 wherein the VH domains of the genetically diverse population of immunoglobulins are biased for one or more VH gene families; 
 wherein expression of each VH domain, which is fused to Fc domains, and each VL domain, which is fused to a constant region, of the immunoglobulins, is effected by a second regulatable promoter; 
 wherein each VH domain, that is fused to an Fc, is in an expression cassette, operably linked to a second regulatable promoter and each VL domain, that is fused to a constant region, is in a separate expression cassette, operably linked to a separate second regulatable promoter; 
 and, wherein the yeast host cells display on their surface immunoglobulins comprising VH domains, fused to Fc domains, and VL domains, fused to a constant regions, wherein the library is created by:
 (i) providing yeast host cells, wherein N-glycans produced by the host cells comprise the structure Man 5 GlcNAc 2 , that express capture moieties comprising cell surface anchoring proteins fused to a moieties capable of binding to an immunoglobulins, wherein the moieties capable of binding to immunoglobulins are members selected from the group consisting of: 
 protein A, a protein A ZZ domain, protein G, protein L and an Fc receptor protein; wherein expression of the capture moieties is effected by a first regulatable promoter; and 
 (ii) transfecting the host cells with said nucleic acid molecules; 
 
 (b) inducing expression of the first regulatable promoter in the host cells for a time sufficient to produce the capture moieties on the surface of the host cells; wherein the first regulatable promoter is inducible without inducing expression of the second regulatable promoter and the second regulatable promoter is inducible without inducing the expression of the first regulatable promoter; and 
 (c) inhibiting expression of the first regulatable promoter and inducing expression of the second regulatable promoters in the host cells, whereby host cells display immunoglobulins at the surfaces thereof having VH domains, that are fused to Fc domains, and VL domains, that are fused to constant regions, and having the antigen binding sites with binding specificity for the antigen of interest. 
 
     
     
       2. The method of  claim 1 , wherein the immunoglobulins comprises synthetic human immunoglobulin VH domains and synthetic human immunoglobulin VL domains and wherein the synthetic human immunoglobulin VH domains and the synthetic human immunoglobulin VL domains comprise framework regions and hypervariable loops, wherein the framework regions and first two hypervariable loops of both the VH domains and VL domains are essentially human germ line, and wherein the VH domains and VL domains have altered CDR3 loops. 
     
     
       3. The method of  claim 2 , wherein in addition to having altered CDR3 loops the human synthetic immunoglobulin VH and VL domains contain mutations in other CDR loops. 
     
     
       4. The method of  claim 2 , wherein each human synthetic immunoglobulin VH domain CDR loop is of random sequence. 
     
     
       5. The method of  claim 2 , wherein human synthetic immunoglobulin VH domain CDR loops are of known canonical structures and incorporate random sequence elements. 
     
     
       6. The method of  claim 1  wherein the method further includes (d) identifying host cells that display an immunoglobulins thereon that have binding specificity for the antigen of interest by contacting the host cells with the antigen of interest and detecting the host cells that have the antigen of interest bound to the immunoglobulins displayed thereon. 
     
     
       7. The method of  claim 1 , wherein the moiety capable of binding to immunoglobulins is protein A. 
     
     
       8. The method of  claim 1 , wherein the cell surface anchoring protein is a GPI protein. 
     
     
       9. The method of  claim 1  wherein the cell surface anchoring protein is a member selected from the group consisting of: alpha-agglutinin, Cwp1p, Cwp2p, Gas1p, Yap3p, Flo1p, Crh2p, Pir1p, Pir4p, Sed1p, Tip1p, Wpip, Hpwp1p, Als3p, and Rbt5p. 
     
     
       10. The method of  claim 1  wherein the first regulatable promoter is a GUT1 promoter or a PCK1 promoter. 
     
     
       11. The method of  claim 1  wherein the second regulatable promoter is a GADPH promoter. 
     
     
       12. The method of  claim 1  wherein the yeast host cell is a member selected from the group consisting of:  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomyces cerevisiae, Saccharomyces, Hansenula polymorphs, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium, Fusarium gramineum, Fusarium venenatum  and  Neurospora crassa.    
     
     
       13. The method of  claim 12  wherein the yeast host cell is  Pichia pastoris.    
     
     
       14. The method of  claim 13  wherein the  Pichia pastoris  cell is genetically engineered to eliminate glycoproteins having alpha-mannosidase-resistant N-glycans. 
     
     
       15. The method of  claim 13  wherein the  Pichia pastoris  cell is genetically engineered to eliminate glycoproteins having phosphomannose residues. 
     
     
       16. The method of  claim 13  wherein the  Pichia pastoris  cell is genetically engineered to produce glycoproteins that have predominantly an N-glycan selected from the group consisting of complex N-glycans, hybrid N-glycans, and high mannose N-glycans. 
     
     
       17. The method of  claim 16  wherein: complex N-glycans are selected from the group consisting of: Man 3 GlcNAc 2 , GlcNAC (1-4) Man 3 GlcNAc 2 , Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 , and NANA (1-4) Gal (1-4) Man 3 GlcNAc 2 ;
 hybrid N-glycans are selected from the group consisting of: Man 5 GlcNAc 2 , GlcNAcMan 5 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 , and NANAGalGlcNAcMan 5 GlcNAc 2 ; and 
 high Mannose N-glycans are selected from the group consisting of: Man 6 GlcNAc 2 , Man 7 GlcNAc 2 , Man 8 GlcNAc 2 , and Man 9 GlcNAc 2 . 
 
     
     
       18. The method of  claim 1  wherein the host cell is  Pichia pastoris , the moiety capable of binding to immunoglobulins is protein A and the cell surface anchoring protein is Sed1p.